1. Field of Invention
The invention relates generally to an apparatus and method for separating molecules on the basis of size and/or structure, and to a method of making the apparatus.
2. Brief Description of Related Technology
It is important in the chemical and biological sciences to be able to separate different molecules from one another. Accurate and precise separation is especially important where the molecules are present in only a small volume solution, such as, for example, in the context of analytical and diagnostic testing. There remains a need to improve the efficiencies of such separations and, thereby, the convenience to researchers working in the chemical and biological sciences.
Generally, molecular separation techniques can include the use of a matrix (or membrane) where molecular transport and filtration occur perpendicular to the surface of the matrix. In such techniques, only those molecules having a precise, pre-determined molecular weight and/or structure pass through the matrix. These separation techniques, however, are limited. For example, biomolecules may not be amenable to separation by such techniques because, for example, they may undesirably react with, or be rendered inactive by, the separating matrix. Even where biomolecules are amenable to these techniques, the separation can be imprecise, inaccurate, and/or difficult to reproduce due to batch-to-batch variations in the manufacture of the matrices. Poor separation efficiency and/or loss of sample volume also can be encountered.
In the biological sciences, gel fractionation or electrophoresis has been found to be a useful technique to separate and identify biomolecules such as, for example, proteins. Generally, in gel electrophoresis, the gel consists of a matrix of entangled polymer chains, intermixed with a buffer solution. A large number of interconnected pores are present within the matrix. A solution of proteins having a net electrical charge are placed in the matrix and travel through the pores under the influence of an electric field. Typically, a charged protein will move towards the pole with a charge opposite to that carried on the protein. The free-solution mobilities of denatured proteins are identical. In the presence of the gel matrix, however, protein mobilities tend to differ because the larger the protein, the more likely it will encounter a physical restriction in the matrix (either between or within the pores), thus retarding the protein's progress through the matrix relative to smaller proteins. The frictional force of the gel material acts as a protein sieve (or, more generally, a molecular sieve) separating the proteins by size. The rate at which a protein migrates through the electric field and gel matrix depends upon, for example, the strength of the field, size and shape of the protein, relative hydrophobicity of the sample in which the protein is present, and on the ionic strength and temperature of the buffer in which the protein is moving. Thus, as smaller proteins should move through the matrix faster than large proteins, the proteins become separated with fast moving bands of small proteins at the front and slow moving bands of larger proteins trailing behind.
One particular type of gel fractionation is two-dimensional (2-D) gel fractionation, which is useful for separating and identifying proteins in a sample by displacement in two dimensions oriented at right angles to one another. Two-dimensional gel fractionation is generally used as a component of proteomics and is a common step used to isolate proteins for further characterization by, for example, mass spectroscopy. This fractionation technique permits component proteins of the sample to separate over a larger area, increasing the resolution of each component protein. IEF (isoelectric focusing) and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) comprise the two dimensions in a 2-D gel fractionation. In a first dimension, IEF fractionates biomolecules on the basis of pl values. In the subsequent, second dimension, SDS-PAGE further fractionates the previously-fractionated biomolecules based on size-charge ratios, which roughly correspond to a fractionation based on molecular weights.
Despite its widespread use, however, 2-D gel fractionation has its limitations. For example, it is not particularly good at resolving proteins or peptides having a low molecular mass as these often migrate through a polyacrylamide gel too rapidly. 2-D gel fractionation also is unsuitable for many proteins, such as hydrophobic proteins, because the proteins often interact with the gel matrix or otherwise undesirably react rendering subsequent analysis of the proteins difficult or impossible. Even when and where the proteins do not undesirably interact or react, it is often difficult to remove them from the gel, thus compromising the quality of any subsequent analysis of the protein. Another particular limitation is that the fractionation takes a long time to perform and requires extensive manual handling and attention, which makes it a long and laborious process requiring a skilled technician or scientist to master and perform. Performing the fractionation is very much an art, requiring much experimentation to find the correct conditions for sample preparation, focusing times, etc. Moreover, it is often difficult to reproduce the exact processing conditions under which multiple gels are made and, therefore, there can be inconsistencies between the various gels. Furthermore, gels can provide only limited resolution, which is often inadequate for certain molecular separation and analytical operations, and are often not re-usable. Still further, the gel material can disadvantageously degrade—polyacrylamide gel is a neurotoxin having a short shelf-life requiring that it be prepared just prior to use, and having properties that vary from batch to batch. Additionally, and given the foregoing limitations, the technique is often inadequate and/or wholly inappropriate for use in an integrated separation and analysis system. Though there have been advances to improve on certain of the foregoing limitations, many of the limitations still remain.
Alternatives to 2-D gel fractionation include techniques that utilize artificial gel media. In contrast to polyacrylamide gels where the sieving matrix is defined by random arrangement of long-chain polymers, the sieving matrix in artificial gels is defined by microfabrication and/or nanofabrication. Thus, the dimensions and topology of the sieving matrix in an artificial gel can be controlled and measured more precisely, and can be mass-produced more easily. For example, conventional photoresist-based lithography can be used to etch a pattern of obstacles on a silicon substrate (floor), which can be sealed with a glass or elastomeric ceiling layer to form a sieve through which a solution of molecules can be electrophoresed. Similarly, monolithic structures can be prepared with a sacrificial layer sandwiched between a dielectric floor and ceiling layers to define a working gap, wherein the sacrificial layer represents what will be the open space in the finished structure and, thus, the negative of the desired pattern of obstacles is etched into it. After the floor, ceiling, and retarding obstacles have been put in place, the sacrificial layer is removed by a wet chemical etch, leaving a working gap whose vertical dimensions are defined by the thickness of the removed layer. Due presumably to critical dimension limitations, however, only nucleic acid separations have been reported with these structures. Protein albumin is about four nanometers (nm) wide and about fifteen nanometers in length and, therefore, is too small to interact physically with patterned structures having 100 nm diameter pillars on a 200 nm pitch. Other techniques contemplate the use of electrochromatography, in situ casting of sieving media within preformed channels of a substrate, and the use of porous materials such as porous silicon as a porous media. Notwithstanding these advances, there remain limitations not adequately addressed in the art.
While the disclosed apparatus and methods are susceptible of embodiments in various forms, there are illustrated in the drawings (and will hereafter be described) specific embodiments of the invention, with the understanding that the disclosure is intended to be illustrative, and is not intended to limit the invention to the specific embodiments described and illustrated herein.